1st Area Medical Laboratory Participation in the FDA Specificity Evaluation of the JBAIDS Influenza A/H5 Detection System

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1st Area Medical Laboratory Participation in the
FDA Specificity Evaluation of the JBAIDS
Influenza A/H5 Detection System

• SSG Edward Loudenclos IV
• NCOIC, Bio/Endemic Disease Section

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AGENDA

• Evaluation Abstract
• Background A/H5 influenza
• Background the JBAIDS analyzer
• H5 Project Overview
• The 1st AML?
• Sample Processing and Testing
• THE EXPERIENCE!
• Results and Summary
• Acknowledgements and References

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Evaluation Abstract

• The possibility of an emerging A/H5 pandemic is
  cause for great concern. Historically, influenza
  pandemics have killed countless millions. Fast
  and early detection impacts treatment and
  quarantine, and is CRITICAL!

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• Real Time PCR is recognized as the standard
  testing method for A/H5 by the CDC.
• The JBAIDS is a deployable Real Time PCR analyzer
  operated by DoD Personnel.
• Idaho Technology Inc. re-optimized the CDC A/H5
  Flu Panel for use on the JBAIDS analyzer.

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BACKGROUNDA/H5 Influenza
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A/H5 Influenza (Avian Flu)

• Primarily infects birds, but can infect humans.
• Infected birds have been reported in at least 40
  different countries.
• Human cases reported in at least 15 different
  countries.
• Mortality rate is approximately 61! Death toll
  world-wide would exceed 1918 pandemic.

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H5 Maps
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• Primary mode of transmission bird to human, but
  possibility of mutation exists for human to human
  transmission.
• Risk factors that promote possible pandemic
• Poor Infrastructure
• Economics
• Lack of surveillance
• Lack of public awareness and education

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• Rapid detection of humans infected with H5
  provides the following benefits
• Patient isolation, prevents possibility of
  additional exposures.
• Early treatment.
• Viral Characterization (did it mutate?!)
• What PCR assays are available now for detecting
  H5?

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• CDC Human Influenza Virus Real-time RT-PCR
  Detection and Characterization panel (rRT-PCR Flu
  Panel).
• Detects several different flu types, including
  A/H5
• Sample extraction and purification can be done
  using different kits (i.e. Qiagen)
• Manual or Automated extraction.

ABI 7500 FAST
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ABI 7500 FAST Real Time PCR using CDC Flu Panel

• Pros

• Cons

• CDC Validated
• FDA Cleared
• Reliable Results
• Sensitivity

• Size
• Not mobile
• Consumables (temperature issues)
• Operator Training

How do we address these problems?
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BACKGROUNDThe JBAIDS Analyzer
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JBAIDSJoint Biological Agent Identification and
Diagnostic Systems

• DoD accepted Platform for both identification and
  diagnostic confirmation of biological agents and
  pathogens of concern.
• Can operate in fixed medical facilities, or
  deployed mobile units.
• Developed by Idaho Technology, Inc.

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JBAIDSSpecifications

• Approximately 36 lbs packed.
• 110/220v power switching capability.
• Rugged, carbon fiber case. 1-meter drop test.
• Meets MILSTD 810F requirements
• Easy user-level maintenance, replacement parts
  issued with instrument.
• 32-sample capacity
• Includes Tough-book laptop and software.
• Real-time PCR for both DNA and RNA.
• Cycle times vary (40 minutes for DNA, 70 minutes
  for RNA due to RT step)

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JBAIDSExtraction

• ITI produces Extraction kits for use with the
  JBAIDS analyzer
• SWIPE Nasal Swabs (DNA), Surface Swabs (DNA and
  RNA), live culture (DNA and RNA), Powder (DNA and
  RNA) Pus swabs, lymph node aspirates.
• VIBE Whole Blood (RNA), Nasal Swabs (RNA),
  Sputum (DNA and RNA).
• SCOOP Stool and Soil samples.
• QFLOW DNA Whole Blood(DNA), Air in PBC (DNA),
  Water, Milk, Salad, Ground Beef, Tuna Salad,
  Gastric Washings.
• QFLOW RNA Air in PBS(RNA).
• PLATINUM PATH All Sample Types, can extract DNA
  and RNA simultaneously (Uses Mag-Bead Technology).

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JBAIDSReagents

• Vaccinia (Orthopox)
• Yersina pestis (Plague)
• Bacillus anthracis (Anthrax)
• Francisella tularensis
• Brucella melitensis
• Rickettsia prowazekii
• Burkholderia spp.
• Variola (Smallpox)
• Coxiella burnetii
• Ebola
• Marburg
• Eastern, Western, and Venezuelan equine
  encephalitis

• FDA Cleared Reagents
• Anthrax IVD
• Plague IVD
• Tularemia IVD
• Kits are freeze-dried, lyophilized reagents. Can
  be stored at Room Temp.

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JBAIDS H5 Project Overview
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Overview Clinical Testing Locations

• 3 Tests Sites

Naval Health Research Center San Diego, CA
Brooke Army Medical Center San Antonio, TX
Tripler Army Medical Center Honolulu, HI
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OverviewSample Criteria

• 600-950 Specimens
• Two sample types
• Throat Swabs
• Nasopharyngeal Swabs
• 0.6mL-1.5mL sample volume
• Specimens previously tested using standard
  diagnostic methods
• De-identified (no Personally Identifiable
  Information)

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OverviewSample Flow
Sick. Flu-like Symptoms
Samples obtained
Lab results negative for Flu
Samples De-identified
Sample aliquots for extraction
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OverviewExtraction

• De-identified Specimens split 3 ways to be
  extracted using the following kits
• IT 1-2-3 VIBE Extraction Kit (on JBAIDS)
• IT 1-2-3 Platinum Path Extraction Kit (on JBAIDS)
• Qiagen QIAamp RNA mini Kit (on ABI 7500 FAST)
• Positive and Negative extraction controls are
  processed with every specimen batch.
• All extracted samples must be sent to analysis
  the day of extraction.

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OverviewResults

• Specimens were tested against 3 targets
• 2 targets specific to A/H5 (Asian Lineage)
• Control target Human RNase P gene (functioned as
  Extraction and Inhibition control)
• ALL results from the processed specimens must
  match regardless of extraction method and PCR
  analyzer used.

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Here comes the1st Area Medical Laboratory!
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How the 1st AML got involved

• Tripler Army Medical Center.
• Small number of people JBAIDS trained.
• Staff had multiple duties.
• Clinical Mission gt Research Mission.
• Time requirements for extraction and testing
  necessitated Full-time staffing.
• Oh, and

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• LTC Hamilton at TAMC submitted request for
  assistance through Pat Craig at JPEO-CBMS.
• CBMS issued request to COL Freund, Commander, 1st
  AML.
• Two Soldiers Selected
• SSG Loudenclos
• SGT Harris
• TDY in HAWAII from 30 NOV 09 23 DEC 09

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Sample Processing and Testing
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Extraction
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Reagent Prep
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Analysis
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Results
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The Hawaiian Experience!
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The Results!
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• Total number of samples evaluated 986
• 518 Throat Swabs
• 468 Nasopharyngeal Swabs
• JBAIDS Influenza A/H5 Detection Kit resulted with
  a Clinical Specificity of 99 and with a 95
  confidence. (This met target goals!)
• All findings, results, and data were submitted to
  the FDA for review on 29 JAN 2010

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Future Goals

• The JBAIDS Program Office is currently working on
  taking the full Influenza test panel (A,B,
  seasonal A/H1, season A/H3, swine A and 2009 A/H1
  assays) through 510(k)
• Anticipated FDA clearance is 2nd or 3rd Quarter
  2011.

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Summary

• A/H5 Avian Flu could pose a grave pandemic
  threat.
• Rapid diagnostic testing necessary to help
  prevent spread of infection.
• Real-time PCR is set as standard for testing.
• ITIs H5 Flu kit can provide same level of
  sensitivity and confidence as currently accepted
  methods.

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Acknowledgements

• Tripler Army Medical Center
• LTC Hamilton, CPT Barrera, Mr. and Miss. Eberly
• Idaho Technology, Inc
• Sean Phipps, Beth Lingenfelter, Sarah Jeffs
• JPEO-CBMS and JBAIDS Program Office
• Pat Craig, James Karaszkiewicz, Jennifer
  McLaughlin
• 1st Area Medical Laboratory
• COL Freund, SGM Krueger, LTC Pell, CPT Pierson

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References

• Beigel, J.H., et al., Avian influenza A (H5N1)
  infection in humans. N Engl J Med, 2005. 353(13)
  p. 1374-85.
• WHO, Avian Influenza Update. http//www.who.int/cs
  r/disease/avian_influenza/en/, 2008.
• CDC, Key Facts About Avian Influenza (Bird Flu)
  and Avian Influenza A (H5N1) Virus.
  http//www.cdc.gov/flu/avian/gen-info/facts.htm,
  2007.

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• For up-to-date information on either H5 and H1
  Influenza
• World Health Organization
• http//www.who.int/en/
• Centers for Disease Control and Prevention
• http//www.cdc.gov/

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QUESTIONS?